The aim of plant biotechnology is the generation of plants with advantageous novel properties, such as pest and disease resistance, resistance to environmental stress (e.g., water-logging, drought, heat, cold, light-intensity, day-length, chemicals, etc.), improved qualities (e.g., high yield of fruit, extended shelf-life, uniform fruit shape and color, higher sugar content, higher vitamins C and A content, lower acidity, etc.), or for the production of certain chemicals or pharmaceuticals (Dunwell 2000). Furthermore resistance against abiotic stress (drought, salt) and/or biotic stress (insects, fungal, nematode infections) can be increased. Crop yield enhancement and yield stability can be achieved by developing genetically engineered plants with desired phenotypes.
For all fields of biotechnology, beside promoter sequences, transcription terminator sequences are a basic prerequisite for the recombinant expression of specific genes. In animal systems, a machinery of transcription termination has been well defined (Zhao et al., 1999; Proudfoot, 1986; Kim et al., 2003; Yonaha and Proudfoot, 2000; Cramer et al., 2001; Kuerstem and Goodwin, 2003). Effective termination of RNA transcription is required to prevent unwanted transcription of trait-unrelated (downstream) sequences, which may interfere with trait performance (see below for more details). Especially arrangement of multiple gene expression cassettes in local proximity (e.g., within one T-DNA) is often causing suppression of gene expression of one or more genes in said construct in comparison to independent insertions (Padidam and Cao, 2001). This is causing problems especially in cases were strong gene expression from all cassettes is required
Previously efficiency of transcription termination had to be analyzed either by in vitro or in vivo transcription analysis of individual transcription termination sequences, which is a laborious and time-consuming procedure based on trial-and-error (Yonaha and Proudfoot, 1999, 2000; Yarnell and Roberts, 1999). To simplify this process, single nucleotide-recognizing probe such as beacon has been used for in vitro transcription (Liu et al., 2002).
In plants, understanding transcription termination and re-initiation is at the infant stage. There are no clearly defined polyadenylation signal sequences. Hasegawa et al. (2003) were not able to identify conserved polyadenylation signal sequences in both in vitro and in vivo systems in Nicotiana sylvestris and to determine the actual length of the primary (non-polyadenylated) transcript. There are vague ideas that weak terminator can generate read-through, which affects the expression of the genes located in neighboring expression cassettes (Padidam and Cao, 2001). Appropriate control of transcription termination will prevent read-through into sequences (e.g., other expression cassettes) localized downstream and will further allow efficient recycling of RNA polymerase II, which will improve gene expression.
Prediction of functional, efficient transcription termination sequences by bioinformatics is not feasible alternative since virtually no conserved sequences exist which would allow for such a prediction. Prediction of the efficiency in transcription termination of such sequences is even more beyond. Furthermore, experimental determination of the actual length and sequence of the primary transcript is difficult since these structures are highly instable being rapidly converted into polyadenylated transcripts (Hasegawa et al., 2003).
Production of genetically modified cells and organisms (such as plants) requires appropriate recombinant DNA in order to introduce genes of interest. The recombinant DNA contains more than one expression cassette, in general. The expression cassette is composed of promoter, gene of interest, and terminator. The expression of the gene of interest in the expression cassette can be negatively affected by inappropriate termination of transcription from the neighboring cassette. Transcriptional read-through and/or multiple use of the same transcription termination sequence may have one or more of the following disadvantages:    1. Unwanted expression of downstream sequences may cause undesirable effects (e.g., changes in metabolic profile, gene silencing etc.).    2. Unwanted expression of downstream sequences raises higher hurdles in deregulation proceedings.    3. Multiple use of identical transcription termination sequences may lead to failure of the whole transgenic expression approach by epigenic silencing. Because the present panel of evaluated transcription termination sequences is currently very limited, multiple use of the same transcription termination sequence in one transgenic organism is often unavoidable, which has proofed to result in unintended silencing of the entire transgenic expression constructs (Matzke 1994; Matzke 1989)    4. Enablement of constructs comprising multiple gene expression cassettes without undesired interaction of transcription of different cassettes. Such interactions may—depending on the orientation of the cassettes—include unintended expression (e.g., in case of expression cassettes having the same direction of their reading frames) or unintended gene silencing (e.g., in case of inverted orientation of the cassettes).
In consequence, there is an unsolved demand (especially in the plant biotech area) for tight and alternative transcription termination sequences. There is no easy and reliable screening system to identify “tight” terminators that efficiently terminate transcription. It is therefore an objective of the present invention, to provide a method to easily identify such termination sequences and to provide tight and alternative transcription termination sequences for use on plants. This objective is achieved by this invention.